The molecular basis for different recognition of substrates by phosphodiesterase families 4 and 10

Phosphodiesterases (PDEs) are key enzymes that control the cellular concentrations of the second messengers cAMP and cGMP. The mechanism for selective recognition of substrates cAMP and cGMP by individual PDE families remains a puzzle. To understand the mechanism for substrate recognition by PDE enzymes, the crystal structure of the catalytic domain of an inactive D201N mutant of PDE4D2 in complex with substrate cAMP has been determined at 1.56 A resolution. The structure shows that Gln369 forms only one hydrogen bond with the adenine of cAMP. This finding provides experimental evidence against the hypothesis of two hydrogen bonds between the invariant glutamine and the substrate cAMP in PDE4, and thus suggests that the widely circulated "glutamine switch" model is unlikely the mechanism for substrate recognition by PDEs. A structure comparison between PDE4D2-cAMP and PDE10A2-cAMP reveals an anti configuration of cAMP in PDE4D2 but syn in PDE10A2, in addition to different contact patterns of cAMP in these two structures. These observations imply that individual PDE families have their characteristic mechanisms for substrate recognition.     

Identification of the novel splicing variants for the hPXR in human livers

Chronic anthracycline administration to rabbits causes impairment of cardiac contractility and decreased gene expression of the calcium-induced calcium release channel of sarcoplasmic reticulum (SR), the ryanodine receptor (RYR2). The C-13 hydroxy metabolite (doxorubicinol), formed in the heart, has been hypothesized to contribute to anthracycline cardiotoxicity. C-13 deoxydoxorubicin is an analog unable to form the C-13 hydroxy metabolite. Therefore, doxorubicin, C-13 deoxydoxorubicin, or saline was administered to rabbits (1 mg/kg iv twice weekly for 8 weeks). Left ventricular fractional shortening (LVFS) was decreased by chronic treatment with doxorubicin (28 +/- 2%; P < 0.05), but not C-13 deoxydoxorubicin (33 +/- 2%) compared to age-matched pair-fed controls. Doxorubicin, but not C-13 deoxydoxorubicin, caused a significant reduction (P < 0.02) in the ratio of RYR2/Ca-Mg ATPase (SERCA2) mRNA levels (0.57 +/- 0.1 vs 1.22 +/- 0.2, respectively) in the left ventricle. This suggests that doxorubicinol may contribute to the downregulation of cardiac RYR2 expression in chronic doxorubicin cardiotoxicity.     

Identification of novel splice variants of the human CD44 gene

The CD44 gene contains 10 variable exons (v1-v10) that can be alternatively spliced to generate hundreds of different CD44 protein isoforms, several of which have been implicated in the metastatic spread of tumour cells. Here, we describe a cryptic splice site, in intron 6 of the human CD44 gene, used during mRNA processing. This cryptic splice site is used in conjunction with variable exon 3, or independently from it in the form of a pseudo-exon of 49 bp, which generates a stop codon by frame shift in the contiguous variable exon downstream. This pseudo-exon has been found inserted immediately 3' to any other variable exon from v4 to v10, in the final CD44 mRNA. The implication of this cryptic splice site in haltering CD44 protein translation is questioned in the context of Nonsense Mediated Decay and the overall regulation of CD44 expression.     

Two human ACAT2 mRNA variants produced by alternative splicing and coding for novel isoenzymes

Acyl coenzyme A：cholesterol acyltransferase 2 （ACAT2） plays an important role in cholesterol absorption. Human ACAT2 is highly expressed in small intestine and fetal liver, but its expression is greatly diminished in adult liver. The full-length human ACAT2 mRNA encodes a protein, designated ACAT2a, with 522 amino acids. We have previously reported the organization of the human ACAT2 gene and the differentiation-dependent promoter activity in intestinal Caco-2 cells. In the current work, two human ACAT2 mRNA variants produced by alternative splicing are cloned and predicted to encode two novel ACAT2 isoforms, named ACAT2b and ACAT2c, with 502 and 379 amino acids, respectively. These mRNA variants differ from ACAT2a mRNA by lack of the exon 4 （ACAT2b mRNA） and exons 4-5 plus 8-9-10 （ACAT2c mRNA）. Significantly, comparable amounts of the alternatively spliced ACAT2 mRNA variants were detected by RT- PCR, and Western blot analysis confirmed the presence of their corresponding proteins in human liver and intestine cells. Furthermore, phosphorylation and enzymatic activity analyses demonstrated that the novel isoenzymes ACAT2b and ACAT2c lacked the phosphorylatable site SLLD, and their enzymatic activities reduced to 25%-35% of that of ACAT2a. These evidences indicate that alternative splicing produces two human ACAT2 mRNA variants that encode the novel ACAT2 isoenzymes. Our findings might help to understand the regulation of the ACAT2 gene expression under certain physiological and pathological conditions.     

Identification and characterization of heart-specific splicing of human neurexin 3 mRNA (NRXN3)

Three neurexin (NRXN) genes are known in humans, each transcribed from two promoters and extensively spliced at five canonical positions, thus generating thousands of isoforms. For NRXN3, only neuronal expression was reported so far. We reported here on the expression of NRXN3 in additional tissues (lung, pancreas, heart, placenta, liver, and kidney) and on the identification and characterization of heart-specific splicing variants of NRXN3. Cardiac isoforms of NRXN3 probably participate in a complex involving dystroglycan and proteins of extracellular matrix, involved in intercellular connections.     

Identification of novel splice variants of the Arabidopsis DCL2 gene

In Arabidopsis thaliana, Dicer-like protein 2 (DCL2) cleaves double-stranded virus RNA, playing an essential role in the RNA interference pathway. Here, we describe three alternative splicing (AS) forms of AtDCL2: in one, both intron 8 and intron 10 are retained in the mRNA, in second only intron 8 is retained and in the third no intron is retained, but there is a deletion of 56 nucleotides at the end of exon 10. These splicing forms are present in stems and leaves at different development stages. AS was also detected in DCL2 of Brassica rapa, where intron 9, but not intron 8 or intron 10, was retained suggesting that AS may be a common phenomenon in cruciferous plant DCL2s. The retained introns and sequence deletions detected in AtDCL2 changed the reading frame and produced premature terminal codons. The AS forms appeared to be substrates of nonsense-mediated decay of mRNA. Fei Yan and Jiejun Peng contributed equally to this work.     

ASD及其在基因选择性剪接检索中的应用

在生物信息学的飞速发展中,与之相应的各种类型的数据库不断涌现,选择性剪接数据库（Alternative Splicing Database）便是其中之一。本文详细介绍了ASD数据库的主要内容及其功能,并在其子数据库AltSplice中检索NGAL基因的选择性剪接,由此为例说明了ASD数据库在基因选择性剪接检索中的应用。     

The mechanism of sex-specific splicing at the doublesex gene is different between Drosophila melanogaster and Bombyx mori

We have previously reported that Bmdsx, a homologue of the sex-determining gene, doublesex (dsx), was found to be sex-specifically expressed in various tissues at larval, pupal, and adult stages in the silkworm, Bombyx mori, and was alternatively spliced to yield male- and female-specific mRNAs. To reveal sex-specific differences in splicing patterns of Bmdsx pre-mRNA, the genomic sequence was determined and compared with male- and female-specific Bmdsx cDNA sequences. The open reading frame (ORF) consisted of five exons. Exons 3 and 4 were specifically incorporated into the female type of Bmdsx mRNA. On the other hand, exon 2 was spliced to exon 5 to produce the male type mRNA of Bmdsx. As in the case of Drosophila dsx, the OD2 domain was separated by a female-specific intron into sex-independent and sex-dependent regions. Sex-specific splicing occurred in equivalent positions in the Drosophila dsx gene. However, unlike Drosophila dsx, the female-specific introns showed no weak 3′ splice sites, and the TRA/TRA-2 binding site related sequences were not found in the female-specific exon, nor even in any other regions of the Bmdsx gene. Moreover, an in vitro splicing reaction consisting of HeLa cell nuclear extracts showed that the female-type of Bmdsx mRNA represented the default splicing. These findings suggest that the structural features of the sex-specific splicing patterns of Bmdsx pre-mRNA are similar to those of Drosophila dsx but the regulation of sex-specific alternative splicing of Bmdsx pre-mRNA is different.     

Alternative splicing variants of human arsenic (+3 oxidation state) methyltransferase

Arsenic (+3 oxidation state) methyltransferase (As3MT) catalyzes the methylation of trivalent arsenic (As(III)) to monomethylarsonate (MMA(V)) and dimethylarsinic acid (DMA(V)), and plays an important role in the detoxification of arsenicals. Here, we report the identification of two splicing variants of the human As3MT gene. One splicing variant was an exon-3 skipping (Δ3) form which produced a premature stop codon, and the other was an exon-4 and -5 skipping (Δ4,5) form which produced a 31.1 kDa As3MT protein. In addition to the full-length mRNA of As3MT, Δ4,5 mRNAs were detected in HepG2, A549, HL60, K562, and HEK293 cells. The methyltransferase activity of the recombinant Δ4,5 As3MT and wild-type As3MT proteins purified from Escherichia coli was determined. Speciation analysis by HPLC–ICP-MS showed a clear peak of MMA(V) after incubation of As(III) with the wild-type As3MT protein, but not with the Δ4,5 As3MT protein. In addition, COS-7 cells transfected with Δ4,5 As3MT cDNA did not convert As(III) to MMA(V) or DMA(V). The lack of methyltransferase activity of Δ4,5 As3MT seems to be related to the deletion of an S-adenosylmethionine-binding site and a critical cysteine residue. These data suggest that the expression pattern of splicing variants of the As3MT gene may affect the capacity for arsenic methylation in cells.     

Characterization of the Isoenzymes of cyclic nucleotide phosphodiesterase in human platelets and the effects of E4021

In extracts of human platelets, three isoenzymes of cyclic nucleotide phosphodiesterase (PDE), namely, PDE2, PDE3, and PDE5, were identified; activities of PDE1 and PDE4 were not detected. In human platelets, the cGMP-hydrolytic activity was about six times higher than the cAMP-hydrolytic activity, and PDE5 and PDE3 are the major phosphodiesterase isoenzymes that hydrolyze cGMP and CAMP, respectively. PDE5 exhibited organ-specific expression in humans, and platelets were among the tissues richest in PDE5. A novel inhibitor of PDE5, sodium 1-[6-chloro-4-(3,4-methylenedioxybenzyl)aminoquinazolin-2-yl] piperidine-4-carboxylate sesquihydrate (E4021), was a potent and highly selective inhibitor of human platelet PDE5. However, E4021 (up to 10 μM) did not inhibit 9,11-epithio-11,12-methano-thromboxane A₂-induced platelet aggregation, in vitro. E4021 plus SIN-1 (3-morpholino-sydnonimine), at concentrations that had little effect individually, inhibited aggregation. These results suggest the unique distribution of phosphodiesterase isoenzymes in human platelets and the PDE5 inhibitors might be useful as a new class of antiplatelet drugs.